PROCESS AND RAW MATERIAL CONTROL STRATEGIES TO MANAGE VARIABILITY IN CHARGE VARIANT SPECIES OF A MONOCLONAL ANTIBODY

摘要

Manufacturing lots of a monoclonal antibody (mAb) produced from a mammalian cell culture process showed increased variability in charge variant species. Root cause investigation uncovered production bioreactor pH and raw material lot-to-lot variability as potential factors affecting the levels of charge variant species. These factors were studied in a qualified 3L scale-down model representative of the large scale manufacturing process. Scale-down experiments confirmed the sensitivity of charge variants to bioreactor pH. Data mining from two manufacturing sites revealed differences in pH equipment and sample handling that contributed to the overall variability. An even greater magnitude of change was observed as a function of basal medium lot-to-lot variability. The variability was compounded due to the presence of hydrolysates in the basal medium. Peptide mapping was performed to check if differences between the samples with different levels of charge variant species could be observed. Results suggested that two different enzymatic modifications, C-terminal lysine clipping and C-terminal leucine amidation, were likely responsible for changes in the charge variants. Subsequently the effect of media components and trace metals such as manganese, copper, zinc and iron, were tested in high throughput scale-down systems using dose response experiments. Follow-up experiments were performed to evaluate the sensitivity of the process to trace metals. In summary, the two main causes of variability of charge species of this mAb were found to be bioreactor pH and trace metal concentration. Characterization of the antibody at the peptide level revealed mechanisms of formation of the charge variant species. We will present strategies for controlling product quality and increasing process robustness at large scale as a result of this work.